Nucleic acid encoding α chain of human IL-11 receptor

ABSTRACT

The present invention relates generally to novel haemopoietin receptors, or components or parts thereof and to a method for cloning genetic sequences encoding same. More particularly, the subject invention is directed to recombinant or synthetic haemopoietin receptors or components or parts thereof. The receptor molecules or components or parts thereof and their genetic sequences of the present invention are useful in the development of a wide range agonists, antagonists and therapeutics and diagnostic reagents based on ligand interaction with its receptor.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.09/532,263, filed Mar. 22, 2000, now U.S. Pat. No. 7,002,000, which is adivisional of application Ser. No. 08/702,665, filed Dec. 20, 1996, nowU.S. Pat. No. 6,274,708.

The present invention relates generally to novel haemopoietin receptors,or components or parts thereof and to a method for cloning geneticsequences encoding same. More particularly, the subject invention isdirected to recombinant or synthetic haemopoietin receptors orcomponents or parts thereof. The receptor molecules or components orparts thereof and their genetic sequences of the present invention areuseful in the development of a wide range of agonists, antagonists andtherapeutics and diagnostic reagents based on ligand interaction withits receptor.

Bibliographic details of the publications numerically referred to inthis specification are collected at the end of the description. SequenceIdentity Numbers (SEQ ID NOs.) for the nucleotide and amino acidsequences referred to in the specification are defined following thebibliography.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

The proliferation, differentiation and function of a wide variety ofcells are controlled by secreted regulators, known as cytokines. Onesuch cytokine is interleukin (IL)-11 which is a functionally pleiotropicmolecule (1,2), initially characterized by its ability to stimulateproliferation of the IL-6-dependent plasmacytoma cell line, T11 65 (3).Other biological actions of IL-11 include induction of multipotentialhaemopoietin progenitor cell proliferation (4,5,6), enhancement ofmegakaryocyte and platelet formation (7,8,9,10), stimulation of acutephase protein synthesis (11) and inhibition of adipocyte lipoproteinlipase activity (12, 13). The diverse and pleiotropic function of IL-11makes it an important haemopoietin molecule to study, especially at thelevel of its interaction with its receptor.

The structure of the IL-11 receptor is not well known. It is known thatneutralizing antibodies to gp130 inhibit IL-11-dependent proliferationof TF-1 cells (14) and, hence, it is likely that gp130 forms part of thereceptor.

Members of the haemopoietin receptor family generally comprise α- andβ-chains (15,16,17). However, until the advent of the present invention,there was no information on the existence of IL-11 receptor chains. Inwork leading up to the present invention, the inventors developed acloning procedure for haemopoietin receptors which does not requireprior knowledge of their ligands. The cloning procedure has beensuccessful in cloning the IL-11 receptor α-chain permitting, for thefirst time, a detailed molecular analysis of the IL-11 receptor. Thepresent invention provides, therefore, a generalized method for cloninghaemopoietin receptors and in particular component chains thereof whichprovides a basis for developing a range of agonists, antagonists,therapeutic and diagnostic agents based on the IL-11 receptor.

Accordingly, one aspect of the present invention provides a geneticmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding a haemopoietin receptor or a mutant, derivative,component, part, fragment, homologue, analogue or a peptide orpolypeptide equivalent thereof wherein said receptor comprises an aminoacid sequence set forth in SEQ ID NO: 1:

-   -   Trp-Ser-Xaa-Trp-Ser,        wherein Xaa is any amino acid.

More particularly, the present invention contemplates a genetic moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding an IL-11 receptor or a mutant, derivative, component,part, fragment, homologue, analogue or a peptide or polypeptideequivalent thereof wherein said receptor comprises an amino acidsequence set forth in SEQ ID NO: 1:

-   -   Trp-Ser-Xaa-Trp-Ser,        wherein Xaa is any amino acid.

Another aspect of the present invention contemplates a method ofidentifying and/or cloning a genetic sequence encoding or complementaryto a sequence encoding a haemopoietin receptor and in particular anIL-11 receptor or a component or part thereof, said method comprisingscreening a source of genetic material with one or more degenerateoligonucleotides designed from the sequence of amino acids comprisingthe sequence set forth in SEQ ID NO: 1:

-   -   Trp-Ser-Xaa-Trp-Ser        wherein Xaa is any amino acid residue.

The sequence defined in SEQ ID NO: 1 has been identified in both α and βchains of haemopoietin receptors and in particular IL-11 receptor.Accordingly, the method of the present invention is applicable to thecloning of genetic sequences encoding an α-chain, a β-chain or acombination of both α- and β-chains such as in a full length receptor.

Preferably, the genetic molecule is of mammalian origin such as but notlimited to humans, livestock animals (e.g. sheep, cows, pigs, goats,horses), laboratory test animals (e.g. mice, rats, guinea pigs),companion animals (e.g. cats, dogs) or captive wild animals. Mostpreferred origins are from humans and murine species (e.g. mice). Thesource of genetic material may be a genomic library or a cDNA libraryobtained from mRNA from a particular cell type such as would not limitto liver cells, bone marrow cells, placenta cells and heptatoma cells. AcDNA library is preferred and may also be an expression library.Furthermore, for the generation of mutants the cDNA library may beprepared by high error rate polymerase chain reaction (PCR) resulting ina mutant library.

The term “screening” includes any convenient means to identify targetclones. For example, colony hybridization may be employed witholigonucleotide probes or if an expression library is prepared,screening may be, for example, enzyme activity or antibodyinteractivity. Terms such as “components”, “parts” or “fragments”include separately an α-chain and a β-chain or parts thereof.Preferably, the “components”, “parts” and “fragments” are functional andmore preferably a functional α- or β-chain.

The genetic molecule may be single or double stranded, linear or closedcircle DNA (e.g. genomic DNA), cDNA or mRNA or combinations thereof. Thegenetic molecule may also include a vector such as an expression vectorcomponent to facilitate expression of the IL-11 receptor geneticsequence.

In a particular aspect, the genetic sequence encodes the α-chain ofIL-11 receptor and in one preferred embodiment is murine IL-11 receptorα-chain encoded by a nucleotide sequence as set forth in SEQ ID NO: 2 orcomprises an amino acid sequence as set forth in SEQ ID NO: 3, orcomprises a part, derivative, fragment, portion, derivative, homologue,analogue or peptide equivalent thereof. In an alternative preferredembodiment, the genetic sequences encodes the α-chain of human IL-11receptor and comprises the nucleotide sequence as set forth in SEQ IDNO: 4 or an amino acid sequence as set forth in SEQ ID NO: 5 orcomprises a part, derivative, fragment, portion, derivative, homologue,analogue or peptide or polypeptide equivalent thereof. Accordingly, thegenetic sequence may include a molecule capable of encoding a fulllength IL-11 receptor or a fragmented portion thereof such as an α-chainor a β-chain whether functional or not or may correspond to a portionthereof characterized by the amino acid sequence Trp-Ser-Xaa-Trp-Serwherein Xaa is any amino acid residue. Additionally, the geneticsequence or part thereof may act as an antisense molecule or moleculesto mRNA encoding the α- or β-chain of the IL-11 receptor. Such antisensemolecules may be useful in genetic therapy or in the rational design ofagonistic or antagonistic agents.

In a related embodiment, there is provided a genetic sequence whichencodes an IL-11 receptor or a component, part or fragment thereofwherein said genetic sequence comprises a sequence of nucleotides towhich SEQ ID NO: 2 or 4 may hybridise under low stringency conditions.In a further related embodiment, the genetic sequence is defined by theability of an oligonucleotide selected from the following list tohybridise thereto:

5′ (A/G)CTCCA(C/T)TC(A/G)CTCCA 3′; (SEQ ID NO:6) 5′(A/G)CTCCA(A/G)TC(A/G)CTCCA 3′; (SEQ ID NO:7) 5′(A/G)CTCCA(N)GC(C/T)CTCCA 3′; (SEQ ID NO:8) 5′ (A/G)CTCCA(N)GG(A/G)CTCCA3′; (SEQ ID NO:9) 5′ (A/G)CTCCA(C/T)TT(A/G)CTCCA 3′; (SEQ ID NO:10)or a complement sequence thereof or a combination thereof.

The present invention extends to the oligonucleotide defined by one ofSEQ ID NOS: 1 to 6 and/or to labeled forms thereof or oligonucleotidestabilized to reduce nuclease-mediated action thereto.

For the purposes of defining the level of stringency, reference canconveniently be made to Sambrook et al (26) which is herein incorporatedby reference where the washing steps at pages 9.52-9.57 are consideredhigh stringency. A low stringency is defined herein as being in 0.1-0.5%w/v SDS at 37-45 C for 2-3 hours. Depending on the source andconcentration of nucleic acid involved in the hybridisation, alternativeconditions of stringency may be employed such as medium stringentconditions which are considered herein to be 0.25-0.5% w/v SDS at ≧45 Cfor 2-3 hours or high stringent conditions as disclosed by Sambrook etal (26).

The present invention is particularly useful for the cloning ofhaemopoietin receptor α- or β-chains, as exemplified by the cloning ofthe IL-11 receptor α-chain (IL-11rα). This is done, however, with theunderstanding that the present invention extends to a method for cloningall haemopoietin receptors including their α- or β-chains. Reference inthe Examples to an α-chain is considered shorthand notation to theentire receptor or various parts thereof, including the α- or β-chain.

In a further embodiment, the genetic sequence is fused to a heterologousgenetic sequence to thereby encode a fusion molecule with, for example,glutathione-S-transferase, a receptor or subunit thereof for IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-9, erythropoietin, thrombopoietin, growthhormone, prolactin, CNTF, G-CSF, GM-CSF, gp130, or the p40 subunit ofIL-12.

The genetic molecule may be single or double stranded, linear or closedcircle DNA (e.g. genomic DNA), cDNA or mRNA or combinations thereof suchas in the form of DNA:RNA hybrids. The genetic molecule may also includea vector such as an expression vector component to facilitate expressionof the IL-11 receptor or its components or parts. In a preferredembodiment, the genetic sequence encodes the α-chain of IL-11 having anamino acid sequence set forth in SEQ ID NO: 3 (murine) or SEQ ID NO: 5(human) or comprises a part, derivative, fragment, portion, component,homologue or analogue of all or a portion thereof. Most preferably, thegenetic sequence comprises a nucleotide sequence as set forth in SEQ IDNO: 2 (murine) or SEQ ID NO: 4 (human) or comprises a part, derivative,fragment, portion, component, homologue or analogue of all or partthereof.

The present invention further contemplates a kit useful for cloning amember of the haemopoietin receptor family or a component or partthereof, said kit comprising in compartmental form a first compartmentadapted to contain at least one species of oligonucleotides having anucleotide sequence based on the amino acid sequence SEQ ID NO: 1:

-   -   Trp-Ser-Xaa-Trp-Ser        wherein Xaa is any amino acid residue, said kit further        optionally comprising one or more other compartments adapted to        contain one or more other species of oligonucleotide based on        SEQ ID NO: 1 and/or reagents required for a hybridisation assay        for haemopoietin receptor genetic sequences. The kit may also be        packaged for same with instructions for use. Preferred        oligonucleotides include but are not limited to SEQ ID NO: 6 to        10.

Yet another aspect of the present invention is directed to a recombinantpolypeptide comprising a sequence of amino acids corresponding to all orpart of a mammalian IL-11 receptor α-chain. Preferably, the mammal is ahuman or a murine species such as a mouse. The polypeptide maycorrespond to a full length α-chain or may be a functional part,fragment or derivative thereof or may be a part, fragment or derivativehaving agonistic or antagonistic properties. In a preferred embodimentthe polypeptide comprises an amino acid sequence as substantially setforth in SEQ ID NO: 3 (murine) or SEQ ID NO: 5 (human) or having atleast about 40%, more preferably at least about 50%, still morepreferably at least about 65%, even still more preferably at least about75-80% and yet even more preferably at least about 90-95% or greatersimilarity to the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.

The polypeptide may have additional amino acid sequences fused theretoincluding GST, another cytokine, a receptor component or gp130. It maybe glycosylated or unglycosylated depending on the cell used to producesame. Accordingly, the polypeptide may be produced in a prokaryotic cell(e.g. E. coli or Bacilli sp) or in a eukaryotic cell (e.g. mammaliancells such as BA/F3 cells [18] yeast cells, insect cells).

Mutants and derivatives of the recombinant polypeptide haemopoietinreceptor properties include amino acid substitutions, deletions and/oradditions. Furthermore, amino acids may be replaced by other amino acidshaving like properties, such as hydrophobicity, hydrophilicity,electronegativity, bulky side chains, interactive and/or functionalgroups and so on.

Amino acid substitutions are typically of single residues; insertionsusually will be of the order of about 1-10 amino acid residues; anddeletions will range from about 1-20 residues. Deletions or insertionspreferably are made in adjacent pairs, i.e: a deletion of 2 residues orinsertion of 2 residues.

The amino acid variants referred to above may readily be made usingpeptide synthetic techniques well known in the art, such as solid phasepeptide synthesis and the like, or by recombinant DNA manipulations.Techniques for making substitution mutations at predetermined sites inDNA having known sequence are well known, for example through M13mutagenesis. The manipulation of DNA sequences to produce variantproteins which manifest as substitutional, insertional or deletionalvariants are well known in the art. Other examples of recombinant orsynthetic mutants and derivatives of the recombinant haemopoietinreceptor polypeptide of the present invention include single or multiplesubstitutions, deletions and/or additions to any molecule associatedwith the ligand such as carbohydrates, lipids and/or proteins orpolypeptides. Naturally occurring or altered glycosylated forms of thesubject ligand are particularly contemplated by the present invention.

Amino acid alterations to the subject polypeptide contemplated hereininclude insertions such as amino acid and/or carboxyl terminal fusionsas well as intra-sequence insertions of single or multiple amino acids.Generally, insertions within the amino acid sequence will be smallerthan amino or carboxyl terminal fusions, of the order of say 1 to 4residues. Insertional amino acid sequence variants are those in whichone or more amino acid residues are introduced into a predetermined sitein the protein. Deletional variants are characterized by the removal ofone or more amino acids from the sequence. Substitutional variants arethose in which at least one residue in the sequence has been removed anda different residue inserted in its place. Such substitutions may bemade in accordance with Table 1:

TABLE 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu;Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr SerThr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

The terms “analogues” and “derivatives” also extend to any functionalchemical equivalent of the ligand characterized by its increasedstability and/or efficacy in vivo or in vitro. The terms “analogue” and“derivatives” further extend to any amino acid derivative of the ligandas described above.

Analogues of the haemopoietin polypeptide receptor contemplated hereininclude, but are not limited to, modifications to side chains,incorporation of unnatural amino acids and/or derivatising the moleculeand the use of crosslinkers and other methods which imposeconformational constraints on the peptides or their analogues. Examplesof side chain modifications contemplated by the present inventioninclude modifications of amino groups such as by reductive alkylation byreaction with an aldehyde followed by reduction with NaBH₄; amidinationwith methylacetimidate; acylation with acetic anhydride; carbamoylationof amino groups with cyanate; trinitrobenzylation of amino groups with2, 4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groupswith succinic anhydride and tetrahydrophthalic anhydride; andpyridoxylation of lysine with pyridoxal-5′-phosphate followed byreduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbomoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringprotein synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides could beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

The present invention, therefore, extends to peptides or polypeptidesand amino acid and/or chemical analogues thereof having the identifyingcharacteristics of the α-chain of IL-11 receptor.

Accordingly, reference herein to the α-chain of the IL-11 receptor or apolypeptide having IL-11 α-chain properties includes the naturallyoccurring molecule, recombinant, synthetic and analogue forms thereofand to any mutants, derivatives and human and non-human homologuesthereof including amino acid and glycosylation variants.

The availability of recombinant IL-11 receptor α-chain and geneticsequences encoding same permits for the first time the development of arange of agonists, antagonists, therapeutics and diagnostics to treat avariety of conditions involving a deficiency of IL-11, an excess amountof IL-11 or aberrant effects of normal endogenous IL-11 levels.Accordingly, the present invention extends to these agonists,antagonists, therapeutics and diagnostics and to compositions,pharmaceutical compositions and agents comprising one or more of same.

The present invention further described by the following non-limitingFigures and/or Examples.

IN THE FIGURES:

FIG. 1 depicts the structure of the IL-11rα cDNA, showing the 5′ and 3′untranslated regions (solid line) and the coding region containing thepredicted signal sequence

 the mature extracellular domain (□), transmembrane domain

 and cytoplasmic domain

 The size and extent of each of the IL-11rα cDNA clones that weresequenced completely are shown below.

FIGS. 2A-2C, joined at match lines A-A, B-B and C-C, show a comparisonof Nr1 with other members of the haemopoietin receptor family; Aminoacid sequence alignment of murine Nr1, the murine IL-6 receptor α-chain,the human CNTF receptor α-chain, the p40 subunit of human IL-12 and themurine GM-CSF receptor α-chain. Alignments were carried out by eye.

FIG. 3 is a photographic representation of reverse transcriptasepolymerase chain analyses of Nr1 mRNA; Cytoplasmic RNA was prepared fromthe following sources; lane 2, 3T3-L1 cells; lane 3, BAd cells; lane 4,UMR-106 cells; lane 5, PC13 cells; lane 6, NFS-60 cells; lane 7, FDCP-1cells; lane 8 32D cells; lane 9, D35 cells; lane 10, M1 cells; lane 11,J774 cells; lane 12 WEHI-3B D-cells; lane 13, human bone marrow; lane14, mouse bone marrow; lane 15, mouse spleen; lane 16, mouse thymus;lane 17, mouse ovary; lane 18, mouse uterus; lane 19, mouse testis; lane20, mouse epididymus; lane 21, mouse brain; lane 22, mouse heart; lane23, mouse kidney; lane 24 mouse thigh muscle; lane 25; mouse liver andlane 26, mouse salivary gland. 1 μg of each RNA sample and a controlcontaining no RNA (lane 1) was subject to reverse transcription, with anidentical reaction performed in the absence of reverse transcriptase. 5%of first strand cDNA reaction was subjected to PCR with primers specificfor Nr1 (upper panel) or the control GAPDH (lower panel). PCR productswere resolved on a 1.0% w/v agarose gel, transferred to nitrocelluloseand hybridised with internal oligonucleotides specific to GAPDH or Nr1.

FIGS. 4A-4D are graphical representations of scatchard analyses ofsaturation isotherms of IL-11 binding to various cell lines; (4A)parental Ba/F3 cells (●), Ba/F3 cells expressing Nr1 (∘), Ba/F3 cellsexpressing Nr1 and the LIF receptor (▪), (4B) Ba/F3 cells expressing theLIF receptor and gp130 (●), Ba/F3 cells expressing Nr1 and gp130 (▪),Ba/F3 cells expressing the Nr1, LIF receptor and gp130 (∘), (4C)parental M1 cells (●), M1 cells expressing Nr1 (∘), and (4D) 3T3-L1cells (▪) were incubated with various concentrations of labeled IL-11 inthe presence of absence of a 10-100-fold excess of unlabelled IL-11.After 18 hours incubation on ice, bound and free IL-11 were separated bycentrifugation through foetal calf serum. Bound and free ¹²⁵I-IL-11 wasquantitated in a γ-counter and the data was depicted as a Scatchardtransformation. In each case data were normalised for cell number andshown as binding to 10⁶ cells.

FIG. 5 shows the molecular specificity of IL-11 binding to various celllines; Ba/F3 cells expressing the designated receptors were incubated in100 μl of medium containing 60,000 cpm (Ba/F3 Nr1) or 6,000 cpm of¹²⁵I-IL-11 (Ba/F3 Nr1/gp130 and Ba/F3 Nr1/gp130/LIF receptor), in thepresence or absence of 20 ng IL-11 or 200 ng of IL-6, LIF, OSM or IL-3.After 18 hours incubation on ice, bound and free IL-11 were separated bycentrifugation through foetal calf serum. Bound and free ¹²⁵I-IL-11 werequantitated in a γ-counter and the amount of binding was expressed as apercentage of that observed in the absence of competitor.

FIG. 6 shows differentiations of M1 cells expressing Nr1 in response toIL-11; 300 parental M1 cells (left panel) or M1 cells expressing Nr1(right panel) were cultured in 1 ml of semi-solid agar with thedesignated concentration of LIF (∘) or IL-11(●). After 7 days, theproportion of colonies containing differentiated cells were determined.

FIG. 7 shows factor dependent proliferation of Ba/F3 cells expressingvarious combinations of Nr1, gp130 and the LIF receptor; Parental Ba/F3cells, Ba/F3 cells expressing Nr1, Ba/F3 cells expressing the Nr1 andthe LIF receptor, Ba/F3 cells expressing LIF receptor and gp130, Ba/F3cells expressing Nr1 and gp130 and Ba/F3 cells expressing Nr1, the LIFreceptor and gp130 were incubated at 200 cells per well in a volume of15 μl, with the designated concentrations of IL-11 (●), IL-3(□) orLIF(∘), or with 3 μg/ml IL-6 and 500 ng/ml soluble IL-6 receptor α-chain(▴). After 48 hours the numbers of viable cells were counted.

FIGS. 8A-8D, joined at match lines A-A, B-B, C-C and D-D provide arepresentation of the composite nucleotide sequence and the predictedamino acid sequence of the human IL-11 receptor a chain. The predictedamino acid sequence is displayed using the conventional single lettercode.

FIG. 9 is a representation of a comparison of the predicted amino acidsequence of the human (H) and the murine (M) IL-11 receptor a chain. Theasterisk symbol indicates identity. The hatch (#) marks represent gapsintroduced to improve the alignment.

FIGS. 10A-10B are photographic representations of a Southern blotdemonstrating cross-species hybridisation of (10A) murine IL-11 receptora chain cDNA probe (445 bp Sph I/Sac I fragment) and (10B) of humanIL-11 receptor a chain cDNA probe (560 bp Pst I/Xba I fragment fromclone #17.1) to human (H) and to murine (M) genomic DNA digested withHind III. Nylon membrane processed under conditions of high stringency(0.2X SSC, 0.1% w/v SDS, 65° C.). Exposure was for 16 hours at −70° C.using intensifying screens.

FIG. 11 is a diagrammatic representation of structure of the humanIL-11rα cDNA, displaying the 5′ and 3′ untranslated region (solid line)and the coding region containing the signal sequence

 the extracellular domain (□), the transmembrane region

 the cytoplasmic portion (▪) and the poly A tail (AAAA). The approximateposition of the conserved cysteine residues (C) and the WSTWS motif isindicated. The size and extent of the four cDNA clones selected foranalysis is shown below. The approximate positions of the introns isindicated (V) as is their size in bp. The length of the clones isdepicted without the introns. The composite cDNA was obtained fromclines #9.1 and #17.1 by ligation at the indicated Pst I site (arrow)and used for expression studies.

FIG. 12 is a diagrammatic representation of scatchard analyses ofsaturation isotherms of human IL-11 binding to M1 cells manipuated toexpress human IL-11rα ( ), M1 cells expressing the murine IL-11rα(●) andparental M1 cells (∘). Cells were incubated with various concentrationsof labeled IL-11 in the presence of 10-100-fold excess of unlabelledIL-11. After 18 hours incubation on ice, bound and free IL-11 wereseparated by centrifugation through FCS. Bound and free labeled IL-11was quantitated n a γ counter and the data was depicted as a Scatchardtransformation. In each case data were normalised for cell number andshown as binding to 10⁶ cells. The amount of non-specific binding wasbetween 0.1 and 1% of the total labeled IL-11 added. High-affinitybinding was seen for M1 cells expressing human IL-11rα (K_(d)=250 pM)and urine IL-11rα (K_(d)=275 pM). Parental M1 cells did not display anyspecific binding.

FIGS. 13A-13D are photographic representations showing morphology ofparental M1 cells and M1 cells manipulated to express the human IL-11receptor a chain (M1/hIL-11rα) and in response to human IL-11 (1000U/ml) and murine LIF (1000 U/ml). Cell morphology was examined after 5days of incubation. FIGS. 13A-13C show parental M1 cells with: normalsaline (Panel a), LIF (Panel b) and IL-11 (Panel c). FIG. 13D isrepresentative of M1/hIL-11rα cells stimulated with IL-11 (X 400).

FIG. 14 is a graphical representation showing proliferation of parentalBa/F3 cells (▴), Ba/F3 cells manipulated to express the human IL-11receptor a chain (Ba/F3+hIL-11rα) and Ba/F3 manipulated to express humanIL-11 receptor a chain along with human gp130 (Ba/F3+hIL-11rα+gp130).Three clonal cell lines (Ba/F3+hIL-11rα) were established (representedby ●) that were unresponsive. Following the expression of the humangp130 molecule, all cell lines were IL-11 responsive (open symbols).Series dilutions of human IL-11 are shown. The results are means oftriplicates from two experiments. All cells proliferated in IL-3.

The following single and three letter abbreviations for amino acidresidues are used in the specification:

Three-letter One-letter Amino Acid Abbreviation Symbol Alanine Ala AArginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidme His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valme Val V Any residue Xaa X

The following abbreviations are adopted in the subject specification:

-   IL-11: Interleukin 11-   IL-11r: IL-11 receptor-   IL-11rα: IL-11 receptor α-chain-   D: Domain-   SD: Sub-domain-   Nr1: IL-11r

EXAMPLE 1 Library Screening

Commercial adult mouse liver cDNA libraries cloned into λgt10 and λZAP(Clonetech, CA, USA and Stratagene, CA, USA) were used to infectEscherichia coli of the strain LE392. Infected bacteria were grown ontwenty 150 mm plates of agar, to give approximately 50,000 plaques perplate. Plaques were then transferred to duplicate 150 mm diameter nylonmembranes (Colony/Plaque Screen™, NEN Research Products, MA, USA),bacteria were lysed and the DNA was fixed by autoclaving at 100° C. for1 min with dry exhaust. The filters were rinsed twice in 0.1% w/v sodiumdodecyl sulfate (SDS), 0.1×SSC(SSC is 150 mM sodium chloride, 15 mMsodium citrate dihydrate) at room temperature and prehybridisedovernight at 37° C. in 6×SSC containing 2 mg/ml bovine serum albumin, 2mg/ml Ficoll, 2 mg/ml polyvinylpryrrolidone, 100 μM ATP, 10 μg/ml tRNA,2 mM sodium pyrophosphate, 2 mg/ml salmon sperm DNA, 0.1% NP-40 and 200μg/ml sodium azide. The pre-hybridisation buffer was removed. An amountof 1.2 μg of the degenerate oligonucleotides for hybridisation (HYB1,HYB2 and HYB3; Table 1) were phosphorylated with T4 polynucleotidekinase using 960 μCi of γ³²P-ATP (Bresatec, S.A., Australia).Unincorporated ATP was separated from the labeled oligonucleotide usinga pre-packed gel filtration column (NAP-S; Pharmacia, Uppsala, SWEDEN).Filters were hybridised overnight at 37° C. in 80 ml of theprehybridisation buffer containing 0.1% w/v SDS, rather than NP40, and10⁶-10⁷ cpm/ml of labeled oligonucleotide. Filters were briefly rinsedtwice at room temperature in 6×SSC, 0.1% v/v SDS, twice for 30 min at45° C. in a shaking waterbath containing 1.5 l of the same buffer andthen briefly in 6×SSC at room temperature. Filters were then blotted dryand exposed to autoradiographic film at −70° C. using intensifyingscreens, for 7-14 days prior to development.

Plaques that appeared positive on orientated duplicate filters werepicked, eluted in 1 ml of 100 mM NaCl, 10 mM MgCl₂, 10 mM Tris.HCl pH7.4containing 0.5% w/v gelatin and 0.5% v/v chloroform and stored at 4° C.After 2 days LE392 cells were infected with the eluate from the primaryplugs and replated for the secondary screen. This process was repeateduntil hybridising plaques were pure.

EXAMPLE 2 Analyses of Positive Plaques

DNA was prepared from positive plaques using Promega Magic Lambda DNAcolumns (Promega Corporation, WI, USA) according to the manufacturer'sinstructions. An amount of 100 ng of DNA from each positivebacteriophage was sequenced using a fmol sequencing kit (PromegaCorporation, WI, USA) with the ³³P-labeled oligonucleotide primersgt10for, gt10rev and either HYB1, HYB2 or HYB3. The products wereresolved on a 6% w/v polyacrylamide gel and the sequence of each clonewas analysed using the Blast database comparison programs and thetranslation function of the Wisconsin suite of programs.

The sequence of one clone (Nr1-AZ-36) contained motifs characteristic ofthe haemopoietin receptor family. Two oligonucleotides, #26 and #60(nucleotides 946-970 and 1005-1034; FIG. 1; Table 2), were designed fromthis sequence and used rescreen the primary filters from the originalliver library and two other adult liver cDNA libraries. The initiallyisolated cDNA clone, Nr1-AZ-36, and four other cDNA clones (Nr1-30.2,30.3, 30.4 and 30.17) were sequenced completely, on both strands, usingthe dideoxy method (18) with the Pharmacia T7 polymerase sequencing kit(Pharmacia, Uppsala, SWEDEN). The sequence of the new receptor wascompared to the EMBL and Genbank database using the FASTA program.Alignments with known cytokine receptors were carried out by eye.

An alternative, quicker method for the analysis of positive plaquesidentified using degenerate oligonucleotides to the WSXWS motif.

Primary positive plaques are identified and picked.

5 μl of primary plaque eluate was used in a polymerase chain reactioncontaining the following: 5 μl 10×PCR buffer with Mg (BoehringerMannheim), 1 μl 10 mM dATP, dCTP, dGTP and dTTP (Promega Corp), 2.5 μlof each primer at 100 μg/ml and 0.5 μl of Taq polymerase (BoehringerMannheim). The primers utilised were those WSXWS primers used inhybridisation in combination with primers specific to theλ-bacteriophage in which the library was cloned. PCR was carried in aPerkin Elmer 9600 machine using the following protocol: 96° C. for 2min, 25 cycles of 96° C. for 30 sec, 55° C. for 30 sec and 72° C. for 2min, 4° C. indefinitely.

20 μl of the PCR was electrophoresed on a 1% w/v agarose gel in TAE. Anyproducts were isolated using GeneClean reagent and sequenced eitherusing ³³P-labeled WSXWS primers with the fmol sequencing kit (PromegaCorp) or unlabelled WSXWS primers and fluoresceinated dideoxynucleotides with an automated sequencer. The sequence is then used tocheck for motifs common to receptors of the haemopoietin family.

TABLE 2 SEQUENCE OF OLIGONUCLEOTIDES Oligonucleotide Sequence SEQ ID NO:HYB1 5′ (A/G)CTCCA(C/T)TC(A/G)CTCCA 3′ SEQ ID NO:6 HYB2 5′(A/G)CTCCA(A/G)TC(A/G)CTCCA 3′ SEQ ID NO:7 HYB3 5′(A/G)CTCCA(N)GC(C/T)CTCCA 3′ SEQ ID NO:8 #26 5′TGGTCCACGGTGGAGCCCATTGGCT 3′ SEQ ID NO:11 #60 5′CCACACGCGGTACGAGTCAGTGCCAGGGAC 3′ SEQ ID NO:12 gt10for 5′AGCAAGTTCAGCCTGGTTAAG 3′ SEQ ID NO:13 gt10rev 5′CTTATGAGTATTTCTTCCAGGGTA 3′ SEQ ID NO:14 #495 5′CCCTTCATTGACCTCAACTACATG 3′ SEQ ID NO:15 #496 5′CATGCCAGTGAGCTTCCCGTTCAG 3′ SEQ ID NO:16 #449 5′GGGTCCTCCAGGGGTCCAGTATGG 3′ SEQ ID NO:17 #285 5′ GGAGGCCTCCAGAGGGT 3′SEQ ID NO:18 #489 5′ CTCCTGTACTTGGAGTCCAGG 3′ SEQ ID NO:19 #741 5′GGAAAGCTGTGGCGTGATGGCCGTGGGGCA 3′ SEQ ID NO:20 30f1 5′GGGCGGAGGCCGCTGGCGGGCG 3′ SEQ ID NO:21 30r1 5′ TTATCAGCTGAAGTTCTCTGGGG3′ SEQ ID NO:22

EXAMPLE 3 Reverse Transcriptase Polymerase Chain Reaction

First strand cDNA synthesis was performed on 1 μg of polyA+ cytoplasmicRNA. Reverse transcription was carried out at 42° C. for 60 min in 20 μlof 50 mM Tris.HCl pH8.3, 20 mM KCl, 10 mM MgCl₂, 5 mM dithiothreitol, 1mM of each dNTP, 20 μg/ml oligo (dT)₁₅ and 12.5 units of AMV reversetranscriptase (Boehringer Mannheim GmbH, Mannheim, Germany). Controlreactions were performed for each RNA sample under identical conditionsexcept that reverse transcriptase was omitted from the reaction. Thereverse transcription reaction mixture was diluted to 100 μl with waterand 5 μl was used for each PCR reaction. PCR reactions were carried outin 50 μl of reaction buffer (Boehringer Mannheim GmbH, Mannheim,Germany) containing 200 μM of each dNTP, 1 μM of each primer and 2.5 Uof Taq polymerase (Boehringer Mannheim GmbH, Mannheim, Germany). Theprimers used for amplification of IL-11 receptor α-chain (Nr1) cDNAwere, from homologY with other members of the haemopoietin receptorfamily, predicted to span at least one intron. These oligonucleotideswere #449 and #285 (nucleotides 133-156 and 677-661; FIG. 1, Table 2),while for amplification of GAPDH cDNA primers #495 and #496 were used(Table 2). PCR was performed for 30 cycles at 94° C. for 2 min, at 60°C. for 2 min and at 72° C. for 3 min in a Perkin Elmer Cetus Thermalcycler (Perkin Elmer Cetus, CA, USA). An aliquot of the reaction mixturewas electrophoresed on a 1.0% w/v agarose gel and DNA was transferred toa zetaprobe membrane. Southern blots were performed as described by Reedand Mann (19). Hybridisation was carried out with end-labeledoligonucleotides (#489 for the IL-11 receptor α-chain and #741 forGAPDH; Table 2).

EXAMPLE 4 Expression Constructs

Nr1-30.3 was used in a PCR with primers 30f1 and 30r1 (Table 2) togenerate a cDNA that contained little 5′ or 3′ untranslated region. ThePCR product was cloned into the BstX I site of pEF-BOS (21) using BstX Iadaptors (Invitrogen, CA, USA). The cDNA insert was sequenced on bothstrands. cDNAs encoding the human LIF receptor and mouse gp130 were alsosubcloned into pEF-BOS. Receptor cDNAs in pEF-BOS were linearized withAat II prior to transfection. pBluescript derivatives containing cDNAsencoding the selectable markers puromycin transferase (pPGKpuropA) andneomycin transferase (pPGKneopA) transcribed from a PGK promoter andwith the β-globin 3′-untranslated region were linearised with Sca I.

EXAMPLE 5 Cell Transfection

Cells were stably transfected by electroporation. Briefly, cells werewashed twice in ice cold PBS and resuspended in PBS at 5×10⁶ per ml. Anamount of 4×10⁶ cells was aliquoted into 0.4 mm electroporation cuvetteswith 20 μg of pEF-BOS with or without Nr1, gp130 or the LIF receptorcloned into the BstX I site and 2 μg of the selectable markers pPGKpuroor pPGKneo. DNA and cells were incubated for 10 minutes on ice andelectroporated at 270 V and 960 μF in a Bio-Rad Gene Pulser (Bio-RadLaboratories, CA, USA). The cells were mixed with 1 ml of culturemedium, centrifuged through 3 ml of FCS and resuspended in 100 ml ofculture medium. Cells were than aliquoted into four 24 well dishes.After two days, selection was commenced by the addition of geneticin toa concentration of 1.2 mg/ml, of puromycin to a concentration of 40μg/ml for M1 cells and 5 μg/ml for Ba/F3 cells. After 10-14 days, clonesof proliferating cells were transferred to flasks and, after expansion,tested for receptor expression.

EXAMPLE 6 Cytokines

Murine IL-3 and IL-11 were purchased from PeproTech (PeproTech, NJ,USA), human LIF and human OSM were produced using the pGEX system,essentially as described (25).

EXAMPLE 7 Biological Assays

The proliferation of Ba/F3 cells in response to cytokines was measuredin Lux60 microwell HL-A plate (Nunc Inc., IL, USA). Cells were washedthree times in DME containing 20% v/v new born calf serum andresuspended at a concentration of 2×10⁴ cells per ml in the same medium.Aliquots of 10 μl of the cell suspension were placed in the culturewells with 5 μl of serial dilutions of purified recombinant IL-3, IL-11or LIF, or IL-6 at 3 μg/ml and soluble IL-6 receptor α-chain at 500ng/ml. After 2 days of incubation at 37° C. in a fully humidifiedincubator containing 10% V/V CO₂ in air, viable cells were counted usingan inverted microscope.

In order to assay the differentiation of M1 cells in response tocytokines, 300 cells were cultured in 35 mm Petri dishes containing 1 mlof DME supplemented with 20% v/v FCS, 0.3% w/v agar and 0.1 ml of serialdilutions of IL-6, IL-11, LIF or OSM. After 7 days culture at 37° C. ina fully humidified atmosphere, containing 10% v/v CO₂ in air, coloniesof M1 cells were counted and classified as differentiated if theycontained dispersed cells or a corona of dispersed cells around atightly packed centre.

EXAMPLE 8 Binding Studies with IL-11

IL-11 was dissolved at a concentration of 100 μg/ml in 50 mM sodiumphosphate, 150 mM NaCl (PBS), 0.02% v/v Tween 20 and 0.02% w/v sodiumazide at pH 7.4. IL-11 was radio-iodinated according to the method ofBolton and Hunter (24). Briefly, 2 μg of IL-11 was incubated with 2 mCiof monoiodinated Bolton-Hunter reagent (New England Nuclear, MA, USA) atroom temperature in 20 μl of 150 mM sodium borate pH 8.5. After twohours the reaction was quenched with 100 μl of 1M glycine in the samebuffer and the labeled protein was separated from unincorporatedBolton-Hunter reagent using a pre-packed Sephadex G-25 column (PD-10;Pharmacia, Uppsala, Sweden) equilibrated in PBS containing 0.02% v/vTween 20 and 0.02% w/v sodium azide. Prior to use the ¹²⁵IL-11 wasdiluted 10-fold with 50 mM Tris HCl pH 7.5, containing 0.02% v/v Tween20 and 0.02% w/v sodium azide and applied to a 250 μl column ofCM-Sepharose CL-4B (Pharmacia, Uppsala, SWEDEN) equilibrated in the samebuffer. The column was washed with 5 ml of equilibration buffer andeluted with sequential 5 ml aliquots of DME containing 10% v/v FCS. Atthis stage the ¹²⁵I was greater than 95% precipitable with coldtrichloroacetic acid. The bindability of the ¹²⁵I-IL-11 preparation wasassessed as previously described (21) and was approximately 80%. Thespecific radioactivity of the ¹²⁵I-IL-11 was approximately 130,000cpm/ng and was determined by self-displacement analysis (22).

Binding studies were performed essentially as previously described (22).Briefly, 5×10⁵-1.5×10⁷ cells in 40 μl RPMI-1640 medium containing 20 mMHepes pH 7.4 and 10% v/v foetal calf serum (RHF), were incubatedovernight on ice, with between 5×10³ and 2×10⁶ cpm of ¹²⁵I-IL-11, withor without a 100-fold excess of unlabelled IL-11. In other experimentsreceptors were saturated with constant amount of ¹²⁵I-IL-11 andincreasing amounts of unlabelled IL-11 or unlabelled IL-3, IL-6, LIF,OSM or G-CSF. Cell associated and free ¹²⁵I-IL-11 were separated byrapid centrifugation through 180 μl of foetal calf serum and quantitatedin a γ-counter.

EXAMPLE 9 Cloning Cytokine Receptors on the Basis of Sequence Similarity

Members of the haemopoietin receptor family exhibit a relatively lowlevel of sequence similarity. One of the features of receptors in thisfamily is the five amino acid motif Trp-Ser-Xaa-Trp-Ser (WSXWS) (15, 16,17). In an attempt to clone novel haemopoietin receptors, 10⁶ plaquesfrom an adult mouse liver cDNA library were screened with degenerateoligonucleotides corresponding to the WSXSW motif. λ-bacteriophageplaques that appeared positive on the duplicate primary filters werepicked, eluted and isolated by two subsequent rounds of plaqueenrichment. DNA from pure hybridising plaques was then sequenced.

The utility of this technique was demonstrated by the identification ofseveral cDNAs encoding the murine LIF receptor, IL-7 receptor, gp130 anda novel sequence that appeared related to members of the haemopoietinreceptor family which is termed herein “Nr1”. The cDNA (Nr1-AZ-36)encoding this novel receptor was sequenced fully and although itcontained a polyadenylation signal and an extensive poly-A tail, it wasclearly truncated at the 5′ end (FIG. 1).

EXAMPLE 10 Isolation of Full Length Nr1 cDNA and Characterisation of theNovel Cytokine Receptor

To isolate a full length Nr1 cDNA, the original library and a secondadult mouse liver cDNA library were screened with oligonucleotides (#26and #60; Table 2) designed from the 5′ end of clone Nr1-AZ-36. EightcDNA clones were isolated and four were sequenced completely (FIG. 1).Analyses of the cDNA sequences revealed an open reading frame of 1296 bpwhich encoded a protein of 432 amino acids in length. The predictedprimary sequence included a potential hydrophobic leader sequence(residues 1-23), extracellular domain with two potential N-linkedglycosylation sites (residues 24-367), transmembrane domain (residues368-393) and short cytoplasmic tail (residues 394-432). The coremolecular weight of the mature receptor has been initially estimated tobe approximately 36,000 daltons.

The extracellular domain contained residues characteristic of aclassical haemopoietin domain (D200; 15) (FIGS. 1 and 2A-2C), includingproline residues preceding each 100 amino acid sub domain (SD100), fourconserved cysteine residues, a series of polar and hydrophobic residues,and a WSXWS motif. The haemopoietin receptor domain of the new receptorwas preceded by an 87 amino acid immunoglobulin-like domain and followedby 37 amino acids before the transmembrane domain. Regarding its overallstructure and its primary sequence (FIGS. 2A-2C), the new receptor wasmost similar to the IL-6 receptor α-chain (24% amino acid identity), theCNTF receptor α-chain (22% amino acid identity) and the p40 subunit ofIL-12 (16% amino acid identity).

EXAMPLE 11 Expression OF Nr1 mRNA

The distribution of Nr1 mRNA expression was analysed by Northern blotand reverse transcriptase polymerase chain reaction (RT-PCR). Among asurvey of polyadenylated RNA from 15 primary tissue samples and 17 celllines, only RNA from the pre-adipocyte cell line 3T3-L1, yielded adetectable hybridising band of approximately 2.0 kb in length on aNorthern blot. This compares to a length of approximately 1650 bp forthe longest Nr1 cDNA isolated and suggests that this clone may not becomplete at the 5′ end.

The low abundance of the Nr1 mRNA suggested from Northern analysesprompted the use of RT-PCR as a more sensitive means of detection. Allsamples contained GAPDH mRNA as judged by RT-PCR (FIG. 3), however only3T3-L1 cells, the stromal line BAd, the embryonic carcinoma cell linePC13 and the factor dependent haemopoietin cell lines FDCP-1 and D35expressed Nr1 mRNA (FIG. 3). A wide range of primary tissues were alsopositive (FIG. 3) including the haemopoietin tissues bone marrow, spleenand thymus as well as the liver, brain, heart, kidney, muscle andsalivary gland. In mRNA samples from several cell lines and tissuestranscripts for Nr1 could not be detected. Such negative results need tobe confirmed using a more quantitative approach to mRNA analysis. Incontrol experiments, PCR was performed on mRNA that had not beensubjected to reverse transcription. In none of these samples was a Nr1product detected.

EXAMPLE 12 Nr1 is a Low Affinity Receptor for IL-11 and Interacts withgp130 to Generate a High Affinity IL-11 Receptor

Given its sequence similarity with the IL-6 and CNTF receptor α-chainsand its expression in 3T3-L1-cells, it was reasoned that Nr1 might be areceptor α-chain which interacts with gp130 and/or the LIF receptor togenerate a high affinity receptor capable of signal transduction. Sinceno receptor α-chains, similar in structure to the IL-6 receptor α-chain,have been described for LIF, OSM and IL-11, these cytokines representattractive candidates for the cognate ligand of Nr1.

To test whether LIF, OSM or IL-11 bound to the new receptor, thefactor-dependent haemopoietin cell line Ba/F3 and the mouse leukaemiccell line M1 were stably transfected with the vector pEF-BOS containingthe cDNA encoding Nr1. Parental M1 cells express the LIF receptor andgp130 and, therefore, bound ¹²⁵I-LIF and ¹²⁵I-OSM. Expression of Nr1 inM1 cells did not result in altered binding of either ¹²⁵I-LIF or¹²⁵I-OSM. In contrast, Ba/F3 cells expressed neither the LIF receptornor gp130 and no binding of ¹²⁵I-LIF and ¹²⁵I-OSM was observed on eitherparental Ba/F3 cells or cells expressing Nr1.

No binding of ¹²⁵I-IL-11 could be detected on parental M1 or Ba/F3 cells(FIGS. 4A & C). Strikingly, however, expression of Nr1 in each cell typeresulted in the ability to bind ¹²⁵I-IL-11 which suggested that Nr1might be the α-chain of the IL-11 receptor. Scatchard transformation ofthe saturation binding isotherms revealed that the affinity of IL-11 forits receptor differed between the two cell types (FIG. 4A versus 4C).Binding of ¹²⁵I-IL-11 to Ba/F3 cells expressing Nr1 was of very lowaffinity. The apparent equilibrium dissociation constant (K_(D)) forthis interaction was estimated to be approximately 10 pM and cellsexpressed an average of between 2,000 and 8,000 receptors at theirsurface (FIG. 4A). M1 cells transfected with a Nr1 cDNA expressed asimilar number of IL-11 receptors (FIG. 4C), however, the affinity ofthe interaction was higher (K_(D)=400-800 pM). The IL-11 receptorsexpressed on M1 cells transfected with Nr1 were similar in affinity tothe receptors expressed naturally on 3T3-L1 cells (FIG. 4D).

One explanation for the generation of low affinity or high affinityreceptors according to the cell type in which Nr1 is expressed, is thatNr1 itself has an intrinsically low affinity for IL-11, but M1 cellsexpress an excess of an additional receptor component required for thegeneration of a high affinity complex. Indirect evidence exists for therole of gp130 in IL-11 receptor signal transduction, since neutralisingantibodies to gp130 inhibited IL-11 induced proliferation of TF-1 cells.In order to test this proposition directly, gp130 and/or the LIFreceptor were expressed in parental Ba/F3 cells or in Ba/F3 cellsexpressing Nr1.

Parental Ba/F3 cells and Ba/F3 cells expressing gp130 and the LIFreceptor, alone or in combination did not bind IL-11 (FIGS. 4A and B).Ba/F3 cells expressing Nr1 and the LIF receptor, bound IL-11 with a verylow affinity that was indistinguishable from cells expressing IL-11receptor α-chain alone (FIG. 4A). In contrast, when gp130 and Nr1 wereco-expressed in Ba/F3 cells, high affinity receptors for IL-11 weregenerated (FIG. 4B). The affinity of these receptors was similar to thatof receptors expressed by 3T3-L1 cells and M1 cells expressing IL-11receptor α-chain (FIG. 4B-D). Expression of the LIF receptor with Nr1and gp130 did not increase the affinity of IL-11 binding (FIG. 4B).

Nr1 appears to be a receptor that is specific for IL-11. The binding of¹²⁵I-IL-11 to Ba/F3 cells expressing Nr1 was competed for by unlabelledIL-11, but not IL-6, LIF, OSM or IL-3 (FIG. 5). A more complex situationexists in cells in which Nr1 is expressed with gp130 and the LIFreceptor. The binding of ¹²⁵I-IL-11 to Ba/F3 cells expressing Nr1 andgp130, was completed for by OSM and unlabelled IL-11 (FIG. 5), whilebinding to Ba/F3 cells expressing Nr1, gp130 and the LIF receptor wascompeted for by LIF, as well as OSM and IL-11 (FIG. 5).

EXAMPLE 13 Co-Expression of IL-11 Receptor α-Chain and gp130 Allows aProliferative and Differentiative Response to IL-11

Many cytokines exert effects upon cell differentiation as well as celldivision. In the absence of differentiative stimuli, colonies ofparental leukaemic M1 cells are tightly packed and are composed ofundifferentiated blast cells. In response to LIF, OSM and IL-6, but notIL-11, M1 colonies grown in semi-solid agar become dispersed because ofthe induction of macrophage differentiation (FIG. 6). In addition, LIF,OSM and IL-6 suppress the clonogenicity of M1 cells resulting in thedevelopment of reduced numbers of colonies. M1 cells expressing theIL-11 receptor α-chain exhibited a normal response to LIF, OSM and IL-6but now differentiated into macrophages when stimulated by IL-11 (FIG.6). As with LIF, IL-6 and OSM, fewer colonies were produced by M1 cellsexpressing Nr1 in the presence of IL-11 than in control cultures andthese colonies contained fewer cells.

The IL-3-dependent haemopoietin cell line Ba/F3 has been used to studythe capacity of a variety of cytokine receptors to transduce aproliferative signal. Ba/F3 cells are absolutely dependent on IL-3 forproliferation, but do not proliferate in response to IL-11, LIF or IL-6.It was determined, therefore, whether expression of Nr1, gp130 and theLIF receptor broadened the spectrum of cytokines to which these cellscould respond. While none of the cell lines examined could proliferatein response to IL-6 alone, each cell line that expressed gp130,irrespective of whether or not other receptors were co-expressed,proliferated in response to a combination of IL-6 and the soluble IL-6receptor α-chain (FIG. 7). Proliferation in response to LIF requiredcoexpression of the LIF receptor and gp130 (FIG. 7), however, thesecells were unable to proliferate in response to IL-11. Likewise, Ba/F3cells expressing Nr1 alone or Nr1 and the LIF receptor were incapable ofresponding to IL-11 (FIG. 7). Response to IL-11 required coexpression ofboth Nr1 and gp130 (FIG. 7). Half-maximal proliferation of these cellsoccurred at an IL-11 concentration of between 20 and 100 pg/ml.Expression of the LIF receptor, in addition to Nr1 and gp130, did notalter this response (FIG. 7).

EXAMPLE 14 Cloning of the Human IL-11rα

In order to determine the feasibility of cloning the human IL-11rα basedon homology with the murine receptor, analysis of murine and humangenomic DNA was carried out using a murine IL-11rα cDNA fragment as aprobe (for method see Example 13). FIG. 10A shows a specific band of 14kb in human DNA, compared with 4.8 kb in the murine DNA, when examinedunder conditions of high hybridisation stringency (0.2×SSC, at 65° C.).

The same murine probe (445 bp Sph I/Sac I fragment) was then used toscreen approximately 10⁶ plaques from five human cDNA libraries. Theseincluded two adult bone marrow libraries (27; Clontech Cat. no. HL1058a)and libraries from the human placenta (Clontech Cat. no. HL1008b), liver(Clontech Cat. no. HL1001a) and a hepatoma cell line (Clontech Cat. no.HL1015b). Positive plaques were isolated and purified by successiverounds of hybridisation-screening (for method see Example 17).Approximately 30 positive clones were obtained from each of the adultbone marrow libraries and the placental library. No positive clones wereidentified from the liver or hepatoma libraries despite the murinereceptor being isolated from this tissue (see previous Examples). Thepositive plaques were also examined using a PCR-based strategy; plaqueeluates were used as templates in a PCR reaction primed with anantisense oligonucleotide encoding the murine WSXWS motif and anappropriate oligonucleotide primer derived from the vector sequence inthe region adjacent to the cloning site. Three clones from a bone marrowlibrary were initially chosen for detailed characterisation. Southernanalysis using a restriction fragment from the human cDNA identifiedequivalent bands to those detected using the murine IL-11rα, thusconfirming the identity of the human cDNA (FIG. 10B). The nucleotidesequence of the insert from each of these clones (#9.1, #4.3, #8.2), wasdetermined in both directions. The insert from clone #9.1 was used togenerate a probe to re-screen the bone marrow cDNA library and resultedin the identification of another unique clone (#17.1, FIG. 11). Thenucleotide sequence of this clone was also determined in bothdirections.

EXAMPLE 15 Sequence Analysis of the Human I-11rα

As depicted in FIG. 11, clones #9.1, and #4.3 were incomplete whileclones #8.2 and #17.1 encompassed the entire coding region. Clones #8.2and #17.1 contained a 287 bp intronic sequence and clones #4.3 and #8.2contained a 254 bp intronic sequence. These sequences were confirmed asintrons by analysis of genomic DNA clones, exhibited typical splicedonor-acceptor sequences and were attributed to incomplete splicing ofmRNA. FIG. 8 shows the composite nucleotide sequence determined from thefour IL-11 rαcDNA clones. The sequence included 127 bp of 5′untranslated region (UTR) that was represented in 3 clones, and a 3′ UTRwith a polyadenylation signal and poly A tail. There was an open readingframe of 1269 bp which was predicted to encode a protein of 423 aminoacids (a.a.). The predicted protein had a potential hydrophobic leadersequence (1-23 a.a.), extracellular region (24-366 a.a.), transmembranedomain (367-392 a.a.) and a cytoplasmic tail (393-423 a.a.). Theextracellular domain contained two possible sites of N-linkedglycosylation (FIG. 8A-D). As with the murine IL-11rα (see previousExamples) and in common with other cytokine receptors (15;28), the humanIL-11rα exhibited an immunoglobulin-like domain and an hemopoietindomain (D200, FIG. 8A-D) in the extracellular region. The latter wascomposed of two subdomains of 100 a.a. (SD100, FIG. 8) and includedproline residues preceding each subdomain, four conserved cysteineresidues, a series of polar and hydrophobic residues an the WSXWS motif.The variable amino acid “S” was identified as theonine in the humanreceptor compared to alanine in the murine equivalent (see previousExamples).

Several differences were noted between clones isolated from the samelibrary. A nucleotide substitution in clone #4.3 (G

C at 944 bp, FIG. 8A-D) resulted in a different amino acid residue (E

Q at 273 a.a., FIG. 8). Clone #4.3 and #17.1 differed from clone #8.2 bya nucleotide substitution (G

A at 1135 bp, FIG. 8A-D) in the coding region with no consequent changein protein. Also, clones #17.1 and #8.2 differed in the 3′ UTR by asingle substitution (A

G at 1658 bp, FIG. 8A-D). These differences were interpreted asrepresenting polymorphisms.

Comparison of the sequences of the murine and human IL-11rα chainsshowed a high degree of homology (FIG. 12). There was overall 85%identity at the nucleic acid level and 84% at the protein level. Thehomology was more evident in the extracellular and transmembrane regionsand less so in the cytoplasmic tail where the human receptor was 8 aminoacids shorter than the murine equivalent. Neither protein contained anidentifiable tyrosine kinase like domain.

EXAMPLE 16 Expression of the Human IL-11 Receptor a Chain Results inSpecific Binding of Human IL-11 and Permits IL-11 Signalling

The murine myeloid leukemic cell line M1 (29) constitutively expressesmurine gp 130 the signalling molecule for LIF, IL-6, OSM and IL-11receptors. In response to LIF, OSM and IL-6, colonies of parental M1cells in semisolid agar become dispersed as cells differentiate intomacrophages and acquire the ability to migrate through agar. Inaddition, there is suppression of clonogenicity leading to reducedcolony numbers. M1 cells manipulated to express the murine IL-11rαdisplayed specific binding of IL-11 and differentiated in response toIL-11 (see previous Examples). The human IL-11rα was expressed in murineM1 cells using the mammalian expression vector pEFBOS (30; Example 15).Binding studies using ¹²⁵I-labeled human IL-11 were carried out to testwhether IL-11 specifically bound to the these cells (see Example 15 formethods). As shown in Table 3, M1 cells manipuated to express the humanIL-11rα (pools #1-#4) demonstrated significant specific binding of humanIL-11. The positive control cells, M1 cells and Ba/F3 cells expressingthe murine IL-11rα and murine gp130 (see previous Examples) also showedhigh level binding. As expected, the parental M1 cells exhibited nodetectable specific binding of IL-11. Scatchard analysis of saturationisotherms of IL-11 binding to M1 cells that expressed human IL-11rαconfirmed high-affinity binding (FIG. 13A-D). The apparent equilibriumdissociation constant (K_(d)) was estimated to be 250 pM. These cellsexpressed an average 3190 receptors at their surface. This result wascomparable to M1 cells expressing murine IL-11rα (K_(d)=275 pM, and 4815receptors/cell) and was attributed to an interaction of the humanIL-11rα with murine gp130.

Table 4 summarises the results of agar culture experiments of M1 cellsthat expressed human IL-11rα and shows their response to LIF and IL-11.As described above, M1 cells expressing the murine IL-11rα displayedclonal suppression and macrophage differentiation in response to IL-11.In contrast, the central parental M1 cells did not respond to IL-11. Thefour pools of M1 cells manipulated to express the human IL-11rα whentreated with IL-11, showed marked suppression of clonogenicity (Table4). In addition, the few colonies that grew in IL-11 displayed adifferentiated phenotype. All cells lines showed the expected responseto LIF.

M1 cells expressing human IL-11rα and control cells were also examinedin suspension cultures to assess macrophage differentiation in responseto IL-11 and LIF (31; 32). Macrophage morphology was assessed after fivedays in culture. As shown in FIG. 13A-D, the majority of the cellsdisplayed a macrophage phenotype following stimulation with IL-11.Similar results were observed with M1 cells expressing the murineIL-11rα, while parental M1 cells did not respond to IL-11. Thus, theseexperiments documented the ability of the isolated human cDNA to encodea functional receptor protein and demonstrated that co-operation betweenthe human IL-11rα and murine gp130 was sufficient for signaltransduction.

To directly address the requirement of gp130 to human IL-11 receptorsignalling, murine Ba/F3 cells were examined. These cells are totallydependent on IL-3 for survival and do not constitutively express gp130.Ba/F3 cells were manipulated to express human IL-11rα and expanded basedon the expression of the co-electroporated puromycin-resistance gene.Three clonal cell lines were established. These were confirmed toexpress human IL-11rα as assessed by binding of radio-labeled humanIL-11, albeit at low level (106; 97; 116; mean specific counts bound per10⁶ cells versus undetectable binding for parental Ba/F3 cells). Asshown in FIG. 14 these cells were unresponsive to IL-11. The human gp130molecule was then expressed in each of these clonal cell lines:cellsthen proliferated in response to IL-11 (FIG. 14). This result confirmedthe expression of the human IL-11rα in Ba/F3 cells and the requirementfor gp130 for proliferation. Parental Ba/F3 cells used as control didnot respond to IL-11 and, as expected, all cells proliferated inresponse to murine IL-3.

EXAMPLE 17 Human Library Screening

The following human cDNA libraries were screened using the abovementioned murine probe:two bone marrow libraries (27; Clontech Cat. no.HL1058a), a placental library (Clontech Cat. no. HL1008b), a liverlibrary (Clontech Cat. no. HL1001a), and a hepatoma cell library(Clontech Cat. no. HL1015b). Approximately 10⁶ plaques from each librarywere lifted onto nitrocellulose membranes and fixed by incubating at 80°C. for 2 hr. under vacuum. The filters were pre-hybridised for 1 hr. andthen hybridised at 65° C. for 16 hr. in a solution containing 2×SSC, 2mg/ml bovine serum albumin, 2 mg/ml ficoll, 2 mg/mlpolyvinylpyrrolidine, 100 μM ATP, 50 μg/ml tRNA, 2 mM sodiumpyrophosphate, 2 mg/ml salmon sperm DNA, 200 μg/ml of sodium azide and1% w/v SDS. The Filters were finally washed for 30 mins. at 65° C. with0.2×SSC, 0.1% SDS. Positive plaques on duplicate filters were isolatedand purified by further rounds of hybridisation screening.

Human clone #91. (FIG. 11) was also labeled and used to probe one humanbone marrow cDNA library. This resulted in clone #17.1.

An amount of 15 μg of human genomic DNA (obtained from peripheral bloodleucocytes) and murine genomic DNA (obtained from the FDCP-1 cell line)was digested to completion with the restriction enzyme Hind III(Boehringer Mannheim, Germany). DNA fragments were separated on an 0.8%w/v agarose gel and transferred with 0.4 M NaOH on to nylon membrane(Gene Screen Plus, Biotechnology Systems, NEN Research Products).

A 445 bp Sph I/Sac I restriction enzyme digest fragment from the murineIL-11rα clone 30.1 (see earlier Examples) and a 560 bp Pst I/Sba Irestriction digest fragment from the human cDNA clone #17.1 were used asprobes. An amount of 100 ng of DNA was labeled using a randomdecanucleotide labelling kit (Braesatec, Adelaide, S.A., Australia). Theincorporated [³²P] ATP was separated from unincorporated label using aNICK column (Pharmacia, Uppsala, Sweden). The membrane was prehybridisedand hybridised at 65° C. overnight in the buffer recommended by themanufacturer. The membrane was finally washed in 0.1% w/v SDS, 0.2×SSC(30 mM sodium chloride, 3 mM tri-sodium citrate) for 30 min. at 65° C.

EXAMPLE 18 Analysis of Human IL-11rα Positive Plaques

Positive plaques isolated using the murine probe were further screenedby a PCR-based strategy. Eluate from pure plaques (5 μl) was used as atemplate in a 50 μl volume PCR reaction using 2.5 U Taq polymerase(Boehringer Mannheim, Germany), the supplied buffer, 200 μM of eachdNTP. The reaction was primed with 250 ng of an anti-senseoligonucleotide primer corresponding to WSXWS motif5′-[(G/A)CTCCA(N)GC(G/A)CTCAA-3′] (SEQ ID NO. 23) and an appropriatevector oligonucleotide primer that flanked the cloned cDNA:T3 and T7promoter primers for pBluescript plasmid, and the appropriate γgt10 andγgt11 forward and reverse primers. Control reactions that lacked thetemplate were also performed. Three plaques (#91., #4.3, #8.2 isolatedfrom a bone marrow library) were selected. The cDNA were sequenced onboth strands using the dideoxy-termination method (18) and the PharmaciaT7 polymerase sequencing kit (Pharmacia, Uppsala, Sweden).

EXAMPLE 19 Human IL-11rα Expression Constructs and Biological Assays

A composite cDNA construct including the entire coding region and thepolyadenylation signal but excluding the intronic sequences was made byligating restriction enzyme digest fragments from #9.1 (Eco RI/Pst Ifragment) and #17.1 (Pst V/Eco RI fragment). The construct was clonedinto the Bst XI site of pEF-BOS (30) using Bst XI adaptors (Invitrogen,San Diego, Calif., USA). It was linearized with Aat II prior toelectroporation into M1 and Ba/F3 cells. pPGKpuropA and pPGKneopA arepBluescript derivatives containing the cDNA encoding puromycintransferase and neomycin transferase and were co-electroporated intocells and used as a selection markers. Human gp130 cloned into pEF-BOSwas electroporated in BaF3 cells manipulated to express the humanIL-11rα.

M1 cells (29) were grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% v/v Fetal Calf Serum (FCS) in 10% v/v CO₂ at 37° C. Ba/F3cells (33) were grown in RPMI-1640 medium containing 10% v/v FCS andWEHI-3B D-conditioned media as a source of IL-3 (34). M1 and Ba/F3 cellsstably expressing the human IL-11rα construct were generated byelectroporation as described above. Cells were co-electroporated withpPGKPuropA. Clones of Ba/F3 expressing human IL-11rα were expanded withpuromycin antibiotic selection and human gp130 was introduced withpPGKneopA. These cells were expanded in G418.

For biological assays, M1 cells (300 per ml) were cultured in DMEM, 20%v/v FCS, 0.3% v/v agar and with human IL-11 (1000 U/ml) or murine LIF(1000 U/ml) or normal saline. Cultures were incubated in humidified airwith 10% v/v CO₂ at 37° C. After 7 days colonies were counted anddifferentiation was assessed using standard criteria (35). In suspensioncultures 1.5×10⁴ M1 cells were cultured in 1.5 ml of DMEM containing 10%v/v FCS and with or without IL-11 (1000 U/ml) or LIF (1000 U/ml) andincubated as above. Differentiation was determined by morphologicalexamination of May-Grunwald Giemsa stained cells: a minimum of 200 cellswas examined.

The proliferation of Ba/F3 cells was measured in a microwell assay asdescribed above. Briefly, 200 cells/well were incubated in 15 μl ofmedia containing the following stimuli: normal saline, murineinterleukin-3 (IL-3) at final concentration 1000 units/ml and seriesdilutions of human IL-11. Viable cells were counted after 48 hours.

Iodination of IL-11 using the Bolton-Hunter reagent and binding studieswith M1 and Ba/F3 cells were performed as previously described above.

EXAMPLE 20 Source of Cytokines

Murine IL-3 and human IL-11 was purchased from Peprotech (Rocky Hill,N.J., USA) and murine LIF and AMRAD Pty. Ltd. (Melbourne, Australia).Human IL-11 used in ligand binding studies was obtained by expression inCOS-M6 cells. Briefly, a cDNA encoding the mature protein for humanIL-11 was obtained by polymerase chain reaction from cDNA derived from ahuman stromal cell line 197/17 (36). The human IL-11 mature codingregion was inserted into pEF/IL3SIG/FLAG which is a pEF-BOS (30) derivedexpression vector containing sequences encoding the murine IL-3 signalsequence followed by the FLAG sequence (Eastman Kodak, CT, USA), andthen expressed in COS-M6 cells resulting in the secretion of abiologically active human IL-11 protein with a N-terminal flag. TheN-terminal flag human IL-11 was purified by affinity chromatography onan anti-FLAG M2 monoclonal antibody column (Eastman Kodak, CT, USA) asrecommended by the manufacturer with peptide elution followed by gelfiltration chromatography on Superdex 75 (Pharmacia, Uppsala, Sweden).The purified protein gave a single band of MW 25,000 on SDSpolyacrylamide gels.

EXAMPLE 21

Since antibodies to the IL-11 receptor a chain were not available tomonitor expression, constructs were engineered to express a solubleversion of the murine IL-11 receptor a chain with an N-terminal FLAGepitope (International Biotechnologies/Eastman Kodak, CT, USA). First aderivative of the mammalian expression vector pEF-BOS was generated sothat it contained DNA encoding the signal sequence of murine IL-3(MVLASSTTSIHTMLLLLLMLFHLGLQASIS) and the FLAG epitope (DYKDDDDK),followed by a unique Xba I cloning site. This vector was namedpEF/IL3SIG/FLAG.

PCR was performed using to amplify DNA fragments encoding theextracellular domain without the transmembrane or cytoplasmic regions(S24 to Q367). The primers used were:

(SEQ ID NO:24) 5′-ATCTTCTAGATCCCCCTGCCCCCAAGCT-3′ (SEQ ID NO:25) 5′ACTTTCTAGATTATTGCTCCAAGGGGTCCCTGTG-3′

The soluble murine IL-11 receptor α chain PCR product was digested withXba I and cloned, in frame, into the XbaI site of pEF/IL3SIG/FLAG toyield pEF-sIL-11rα.

In order to confirm soluble murine IL-11 receptor α chain could beproduced using the expression vectors pEF-SIL-11rα, COS cells weretransiently transfected with these constructs. Briefly, COS cells from aconfluent 175 cm² tissue culture flask were resuspended in PBS andelectroporated (BioRad Gene pulser; 500 μF, 300 V.) with 20 μg of uncutpEF-sIL-11rα in a 0.4 cm cuvette (BioRad). After 2 to 3 days at 37° C.in a fully humidified incubator containing 10% v/v CO₂ in air cells wereused for analyses of protein expression. Conditioned medium wascollected by centrifugation and stored sterile at 4° C.

Medium was then chromatographed on an anti-FLAG antibody affinity column(International Biotechnologies/Eastman Kodak, CT, USA). Proteins thatfailed to bind to the column were washed through with PBS containing,while those proteins the murine IL-11 receptor α chain proteins whichbound to the column was eluted with 8 ml of ug/ml FLAG peptide. Thepurified soluble murine IL-11 receptor α chain was electrophoresed on aSDS-polyacrylamide gel, which was stained with silver to reveal thepresence of a major band with an apparent molecular weight ofapproximately 40,000 similar to the predicted size of the soluble murineIL-11 receptor α chain.

The purified soluble murine IL-11 receptor α chain was tested for itsability to stimulate the differentiation of M1 cells in the presence orabsence of IL-11. IL-11 and the soluble murine IL-11 receptor α chainwere unable to stimulate M1 differentiation alone, however, whencombined, differentiation was observed in both liquid and semi-solidculture. These results demonstrate that soluble murine IL-11 receptor αchain may act as an agonist, allowing IL-11 to exert effects on cellsexpressing gp130 in the absence of membrane bound IL-11 receptor αchain. In this way soluble IL-11 receptor α chain is similar to solubleIL-6 receptor α chain.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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1. An isolated nucleic acid encoding an IL-11 receptor α chain whichcomprises the amino acid sequence having at least 95% identity with SEQID NO: 5, wherein said amino acid sequence comprises Trp-Ser-Xaa-Trp-Ser(SEQ ID NO: 1) and wherein said IL-11 receptor α a chain binds to IL-11.2. An isolated nucleic acid encoding an IL-11 receptor α a chain whichcomprises Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 1), wherein said nucleic acidcomprises the nucleotide sequence that hybridizes to the complement ofSEQ ID NO: 4 under high stringency conditions comprising hybridizationat 65° C and washing in 0.2X SSC, 0.1% w/v SDS at 65° C. and whereinsaid IL-11 receptor α chain binds to IL-11.
 3. A recombinant vectorcomprising the nucleic acid according to claim 1 or 2.